Sullivan first proposed in 1981 that reduction in body iron stores may protect against cardiovascular disease. Blood donation is known to reduce iron stores and has been tentatively associated with reduced cardiovascular risk in observational studies, but the mechanisms to account for this finding are not well characterized. Our preliminary data demonstrate that high-frequency blood donation is associated with severe reductions in iron stores, decreased oxidative stress, and improved vascular endothelial function when compared with sporadic donation. Since improved vascular endothelial function is known to be associated with decreased cardiovascular morbidity and mortality, these preliminary data support a potential mechanistic link between blood donation and reduced cardiovascular risk. We now propose a prospective interventional trial to investigate potential mechanisms linking blood donation and vascular endothelial function in voluntary blood donors. We hypothesize that serial phlebotomy is associated with improved vascular function related to physiological and biochemical effects of iron removal and blood removal. To test this hypothesis, 150 iron-replete voluntary blood donors currently deferred from active donation for non-medical reasons will be randomly assigned to 3 treatment groups: four 500 ml phlebotomy procedures over 6 months without replacement of lost iron, four 500 ml phlebotomy procedures over 6 months with intravenous replacement of lost iron, or sham phlebotomy. This 3-arm study design provides the means to distinguish the relative contributions of blood loss vs. iron loss on biological responses to serial phlebotomy. Flow-mediated dilation in the brachial artery (a non-invasive measure of vascular endothelial function) in the basal state and in response to transient hyperhomocysteinemia, serum and urinary measures of iron stores and oxidative stress, red cell and plasma volume, regional arterial wall shear stress, and erythropoietin levels will be obtained before and after the assigned study intervention. Mulitvariate mixed effects models will be used to determine the association between study treatment and vascular function and whether physiological and biochemical correlates of iron and blood removal are mediators of this association. This study will provide new insight into the relationship between iron stores and vascular function and will provide new data on the health effects of blood donation that could impact the nation's blood supply. More than 50% of all Americans will require a blood donation at least one time in their lives. We propose a prospective randomized double-blind study to determine the effects of blood donation on vascular function. The overall goal of the proposal is to better characterize the potential impact of blood donation on cardiovascular health. The findings of this study will provide novel data that could favorably impact the nation's blood supply, and could influence public policy decisions on screening for hemochromatosis (one of the most common genetic diseases in the US) and regulation of iron content in nutritional supplements and foods.